Chimeric proteins and methods of immunotherapy

Abstract

The present disclosure provides systems for immune cell regulation and methods of immunotherapy. Systems of the present disclosure for immune cell regulation comprise a chimeric receptor polypeptide, a chimeric adaptor polypeptide, a gene modulating polypeptide (GMP), and a cleavage moiety.

What is claimed is: 
     
         1 . A method of inducing death of a target cell, comprising:
 (a) expressing a system in a lymphocyte; and   (b) contacting said target cell with the lymphocyte under conditions that induce said death of the target cell,   wherein the system expressed in the lymphocyte comprises:
 (i) a chimeric transmembrane receptor polypeptide comprising a ligand binding domain, an immune cell signaling domain, and a gene modulating polypeptide (GMP), wherein the GMP comprises an actuator moiety linked to a cleavage recognition site; and 
 (ii) a chimeric adaptor polypeptide comprising a receptor binding moiety linked to a cleavage moiety, wherein the cleavage moiety is capable of cleaving the cleavage recognition site on the receptor when the receptor binding moiety binds the receptor polypeptide in response to binding of the ligand binding domain to a ligand; 
   wherein, upon contacting the target cell to the lymphocyte, the ligand binding domain binds a ligand present on the target cell to (i) activate cytotoxicity of the lymphocyte, and (ii) release the actuator moiety from the GMP via cleavage by the cleavage moiety at the cleavage site, wherein the actuator moiety in turn modulates expression and/or activity of an immune regulatory protein that enhances lymphocyte cytotoxicity and/or reduces a side effect of lymphocyte activation.   
     
     
         2 . The method of  claim 1 , wherein modulation of expression and/or activity of the immune regulatory protein enhances cytotoxicity of the lymphocyte compared to a system in which the immune regulatory protein expression and/or activity is not modulated. 
     
     
         3 . The method of  claim 1 , wherein modulation of expression and/or activity of the immune regulatory protein reduces a side effect of lymphocyte activation compared to a system in which the immune regulatory protein expression and/or activity is not modulated, wherein the side effect is hypercytokinemia. 
     
     
         4 . The method of  claim 1 , wherein the actuator moiety up-regulates expression and/or activity of the immune regulatory protein. 
     
     
         5 . The method of  claim 1 , wherein the actuator moiety down-regulates expression and/or activity of the immune regulatory protein. 
     
     
         6 . The method of  claim 1 , wherein the immune regulatory is selected from A2AR, B7.1, B7-H3/CD276, B7-H4/B7S1/B7x/Vtcn1, B7-H6, BTLA/CD272, CCR4, CD122, 4-1BB/CD137, CD27, CD28, CD40, CD47, CD70, CISH, CTLA-4/CD152, DR3, GITR, ICOS/CD278, IDO, KIR, LAG-3, OX40/CD134, PD-1/CD279, PD2, PD-L1, PD-L2, TIM-3, and VISTA/Diesl/Gi24/PD-1H (C10orf54). 
     
     
         7 . The method of  claim 6 , wherein the immune regulatory protein is PD-1 or CTLA-4. 
     
     
         8 . The method of  claim 1 , wherein the immune regulatory protein is an immune checkpoint receptor. 
     
     
         9 . The method of  claim 1 , wherein the target cell is a cancer cell. 
     
     
         10 . The method of  claim 9 , wherein the cancer cell is of hematopoietic lineage or a B-cell lymphoma cell. 
     
     
         11 . The method of  claim 9 , wherein the lymphocyte exhibits an enhanced ability to induce death of the target cell as compared to that of a control lymphocyte, wherein the enhanced ability to induce death of the target cell is at least a 1.5-fold increase in induced cell death. 
     
     
         12 . The method of  claim 9 , wherein the cancer cell expresses a ligand indicative of a B-cell lymphoma. 
     
     
         13 . The method of  claim 1 , wherein the ligand is CD19. 
     
     
         14 . The method of  claim 1 , wherein upon binding of the ligand binding domain to the ligand, the receptor undergoes a receptor modification comprising phosphorylation. 
     
     
         15 . The method of  claim 14 , wherein the receptor modification comprises phosphorylation at multiple modification sites, and wherein each modification site is effective to bind a chimeric adaptor polypeptide. 
     
     
         16 . A system capable of inducing death of a target cell, comprising:
 (a) a chimeric transmembrane receptor polypeptide comprising a ligand binding domain, an immune cell signaling domain, and a gene modulating polypeptide (GMP), the GMP comprising an actuator moiety linked to a cleavage recognition site; and   (b) a chimeric adaptor polypeptide comprising a receptor binding moiety linked to a cleavage moiety capable of cleaving the cleavage recognition site when in proximity to the cleavage recognition site,   wherein upon ligand binding to the ligand binding domain, the immune cell signaling domain activates cytotoxicity of a lymphocyte and the adaptor polypeptide binds the receptor polypeptide to effect release of the actuator moiety by cleavage of the cleavage recognition site by the cleavage moiety, the released actuator moiety exhibiting the ability to modulate expression and/or activity of an immune regulatory protein within the lymphocyte.   
     
     
         17 . The system of  claim 16 , wherein the ligand binding domain comprises a single-chain variable fragment (scFv). 
     
     
         18 . The system of  claim 16 , wherein the ligand binding domain binds CD19 or a fragment thereof. 
     
     
         19 . The system of  claim 16 , wherein the immune cell signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). 
     
     
         20 . The system of  claim 16 , wherein the actuator moiety comprises a CRISPR-associated polypeptide (Cas), a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a recombinase, a flippase, a transposase, or an Argonaute protein. 
     
     
         21 . The system of  claim 20 , wherein the actuator moiety comprises a Cas protein that forms a complex with a guide RNA (gRNA). 
     
     
         22 . The system of  claim 21 , wherein the Cas protein is Cas9, Cpf1, or C2c2. 
     
     
         23 . The system of  claim 21 , wherein the Cas protein substantially lacks DNA cleavage activity. 
     
     
         24 . The system of  claim 21 , wherein the Cas protein is linked to a transcriptional repressor or a transcriptional activator. 
     
     
         25 . The system of  claim 16 , wherein the cleavage recognition site comprises a polypeptide sequence and the cleavage moiety comprises a protease. 
     
     
         26 . The system of  claim 16 , wherein the cleavage moiety is a TEV protease, and wherein the cleavage recognition site is a TEV protease cleavage site. 
     
     
         27 . A lymphocyte expressing the system of  claim 16 . 
     
     
         28 . A population of lymphocyte cells, individual lymphocyte cells expressing the system of  claim 16 , wherein said population of lymphocyte cells is characterized in that: upon exposing said population of lymphocyte cells to a target cell population in an in vitro cell death assay, the population of lymphocyte cells induces death of at least 45% of the target cells within about 2 days when the ratio of the number of lymphocyte cells in said population of lymphocyte cells to the number of target cells in the target cell population is about 1:2 or less.